Since the 1990s, genome projects for various organisms have been conducted, and the entire genomic nucleotide sequence has already been determined in many species of organism, including humans. Currently, post-genomic studies such as the functional analyses of proteins encoded by individual genes are eagerly being conducted using the results of the genome analyses. In particular, studies using genetically modified animals are essential in analyzing how a particular gene functions in the living body at the individual level, and they have gained an important position since new therapeutic agents can be developed by using genetically modified animals as disease model animals. Therefore, many genetically modified animals such as transgenic animals, in which particular exogenous genes have been introduced, and gene-knockout animals, in which particular endogenous genes have been disrupted, have been developed.
Recently, much attention has been focused on genome editing as a technique for knocking out target genes in various species of organism. Genome editing is a technique for introducing a mutation at a particular site into a target gene by using an artificial nuclease (e.g., ZFN or TALEN) or an RNA-directed nuclease (e.g., CRISPR/Cas) that can recognize and cleave any target sequence in a DNA chain (Non-Patent Document 1). The artificial nuclease such as ZFN or TALEN is a chimeric protein in which a DNA binding domain that specifically recognizes a target sequence and a DNA cleavage domain of the restriction enzyme FokI are linked, whereas, in the CRISPR/Cas system, a small RNA molecule, referred to as the guide RNA (gRNA), recognizes a target sequence and the RNA dependent DNA nuclease Cas9 cleaves the DNA. Since designing and synthesis of RNA that specifically recognizes a target sequence are much easier than those of protein, the CRISPR/Cas system is an increasingly attractive genome editing tool.
Molecules that can specifically recognize and bind to a DNA sequence include peptide nucleic acids (PNAs), in addition to DNA and RNA. PNAs are unnatural nucleic acid analogs that are artificially produced to have properties similar to DNA and RNA, and have a peptide backbone that replaces the deoxyribose-phosphate backbone in DNA. Advantages of PNA are, for example, the following. (1) The PNA/DNA double-strand or the PNA/RNA double-strand can be formed stably without being affected by pH or salt concentration because PNAs have no negative charges derived from phosphate groups present in DNA and RNA and are neutral. (2) PNAs have extremely high specificity of nucleotide sequence recognition because the decrease in the Tm value of PNA is greater than those in DNA or RNA. (3) PNAs are suitable for use in cells because they are highly resistant to the degradation with nucleases and proteases in vivo. Therefore, various PNAs having a peptide backbone have been developed (Non-Patent Documents 2 to 7).
Focusing on the above advantages of the PNA, genome editing using PNA has also been attempted. For example, Komiyama et al. at the University of Tokyo have succeeded in disrupting a target gene by introducing a PNA that specifically binds to the target gene and a cerium ion into cells by electroporation (Non-Patent Document 8). In this method, the double strand of DNA at a target site is unwound by intercalating PNA oligomers into the double-stranded DNA adjacent to the target site, and the single-stranded target site is cut with using a cerium ion. PNAs have extremely high specificity of nucleotide sequence recognition and therefore they are specifically inserted at the target site. However, a cerium ion may cut single-stranded DNAs other than the single-stranded DNA formed by the insertion of the PNAs, resulting in a problem of poor specificity. Thus, there are high expectations for artificial nucleases having high specificity for any target sequence as a useful genome editing tool, by fusing a nuclease domain consisting of a protein and a target DNA binding domain consisting of PNAs.
However, all PNAs have been produced by chemical synthesis thus far. The synthesis of PNA oligomers is conducted by solid-phase synthesis similar to the peptide synthesis by repeating the condensation of monomers protected with Boc, Cbz, Fmoc, or the like and deprotection. Therefore, in the solid-phase synthesis, the yield of 10-mer PNA oligomers would be as low as about 35% even if the efficiency of one condensation reaction was 90%, and thus the efficient production of long chain PNAs is extremely difficult. Furthermore, when chemically synthesized PNAs are fused with a protein, the problem is that the fusion with PNAs may not be sufficient or excessive PNAs are fused, depending on the nature of the protein. Alternatively, a PNA and a protein can be continuously synthesized by a chemical method; however, enzyme proteins such as nucleases are usually composed of 100 amino acids or more, and it is therefore substantially impossible to synthesize enzyme proteins at a practical level in consideration of the problem of reaction efficiency in the solid-phase synthesis method as described above. Therefore, establishing a process that makes it possible to synthesize a PNA-protein fusion by a process other than chemical synthesis is expected to provide a big advantage in post-genomic studies.
Puromycin, blasticidin, and the like, are known as naturally occurring NBAs or analogs thereof, and they are used as antibiotics because they inhibit the process of translation and inhibit the protein synthesis by binding to the ribosome. Therefore, it has been considered to be difficult to synthesize proteins introduced with PNAs containing NBAs as constituent units by a ribosomal translation system.